1. Introduction
Global warming is making rapid and transformative changes in the traits associated with viticulture across the world [
1]. The majority of Mediterranean wine regions are known to confront very hot summers and mild winters, with peak temperatures often surpassing the threshold of 40 °C in the growing season [
1,
2,
3,
4,
5]. The heightened severity and frequency of several summer-induced stresses exerted in the vineyard are one of the most common ramifications of warming trends [
1]. They encompass the simultaneous emergence of heat and water stress in conjunction with excessive radiation loads, thus leading to necrosis and leaf photoinhibitions, a significant reduction in yields, spoilage of fruit quality, and, particularly in young plantings, vineyard loss [
1,
6].
Indubitably, adopting a drought-resistant rootstock denotes a fundamental choice of establishing a new vineyard when dealing with sites characterized by water scarcity. Even though rootstock breeding, as well as selection programs, have witnessed slow progress in viticulture, as evidenced in a subdued rate of material released over the past century, the pressure associated with warming trends has resulted in the selection of genotypes that can possibly confer to the scion a higher tolerance to abiotic stresses. It is notable that the M4 rootstock ((
Vitis vinifera ×
Vitis berlandieri) ×
Vitis berlandieri cv. Resseguier n.1) has recently been recognized as a promising material to establish vineyards in areas that are vulnerable to summer-related droughts. In that context, Meggio et al. [
7] delineated some physiological attributes of own-rooted M4 vines impacted by water deficit, observing that the physiological performances of the new genotype were better as compared to own-rooted 101−14 Millardet et De Grasset (
Vitis riparia × Vitis rupestris) vines, operating at 30% of field capacity. In addition, M4 demonstrated accelerated recovery upon re-watering [
7]. According to Galbignani et al. [
8], Sangiovese vines grafted to M4 exhibit a slower response to water-stress with regard to deferred pre-dawn water potential as well as whole canopy assimilation drop in comparison to the vines grafted onto SO4 (
Vitis berlandieri × Vitis riparia), which is particularly vulnerable to drought. Moreover, it was observed that the hydraulic conductance rates of M4 were higher than SO4 [
8]. Similarly, while undertaking a comparison of the same rootstock/scion combinations, Merli et al. [
9] demonstrated that Sangiovese grafted onto M4 was able to maintain a higher whole canopy water use efficiency under water stress.
Meanwhile, other studies have directed their attention on M4 rootstock metabolomic, transcriptomic, or proteomic profiling. In an exhaustive transcriptomic evaluation of M4 and 101.14 Millardet et De Grasset roots, Corso et al. [
10] observed that heightened drought tolerance demonstrated by M4 pertained to different modulations amidst water-stress conditions associated with the phenil–propanoid pathway. Correspondingly, while making a comparison between the metabolic and proteomic profile of the two same rootstocks under water stress conditions, Prinsi et al. [
11] highlighted the M4 downregulation of heat shock proteins and abscisic acid linked with the upregulation of metabolites that are osmotically active.
Despite compelling evidence of the positive impacts ascribed to M4 rootstock on the scion’s water status even under extreme water paucity, extant literature provides little information concerning M4 behavior in comparison to other rootstocks that are classified as “drought-tolerant.” As a matter of fact, M4 has primarily been tested in comparison to SO4 [
8,
9] and 101−14 Millardet et De Grasset [
7,
10,
11], two conventional rootstocks that are known to display poor tolerance to water shortage [
12,
13]. In hot or arid conditions, vineyards are typically known to adopt drought-tolerant and robust rootstocks (
Vitis berlandieri × Vitis rupestris) whose performance is good in forming tap roots that are capable of digging water from deep soil layers, including 110 Richter, 1103 Paulsen, or 140 Ruggeri [
12,
13,
14,
15]; for this reason, increasing more hints about directly comparing these rootstocks with M4 would also help facilitate the assessment of marginal gain achieved after the selection of M4. In extant literature [
16], only one paper was able to offer data about a
Vitis vinifera cultivar (Cabernet Sauvignon) that was grafted to M4 as well as to a rootstock that was drought tolerant (namely, 1103 Paulsen), thus suggesting that M4 was able to better sustain berry growth and expedited ripening. Unfortunately, this study excluded any irrigation management, soil water content, or data concerning the status of plant water or physiological parameters.
Introduction of the iso/anisohydry nomenclature to categorize
Vitis vinifera L. varieties dates back to the work by Berger-Landefeldt [
17]; since then, several definitions for the two categories have been proposed. Those attributing to isohydric plants the capacity to maintain a relatively constant leaf water potential (Ψ
leaf) despite changes in soil water potential (Ψ
soil) and vapor pressure deficit (VPD), and ascribing to anisohydric plants the property lo let Ψ
leaf to co-vary more strongly with Ψ
soil and VPD, are the most shared. However, with additional information involving a larger number of varieties, growing conditions (i.e., pot vs. open field) and type of water stress (fast according to a dry-down mode or slow as it might occur in the field under progressive soil drying) led to the general opinion that relative iso/anisohydry is not a dichotomy, rather yet a continuum. In other words, there is considerable variation in water status regulation between the two extremes, and environmental conditions play an important role in affecting such variations. Against the theory of considering iso/anisohydry as a “simple” plant trait, some authors [
18,
19] have even suggested abandoning the concept while hinting to a safer wording such as vulnerable/tolerant genotypes. Yet, the debate does not seem to be exhausted as Ratzmann et al. [
20] have recently countered that iso/anisohydry is still a useful concept, suggesting that the leaf turgor loss point can be a reliable proxy to integrate the complex interactions between plant hydraulic traits. [
21,
22,
23,
24,
25,
26,
27,
28]. According to the limited information available, Grechetto Gentile stands for a near-isohydric behavior, and M4 rootstock has thus far only been tested on near-anisohydric scions [
8,
9,
16,
29].
In this context, this study specifically aims at attaining the following objectives: (a) determine if grafting cv. Grechetto Gentile vine on M4 significantly alters leaf gas exchange and water status vs. 1109 grafted vines during a dry down period followed by re-watering; (b) infer mechanisms that eventually drive different vine performance, according to the used rootstock.
2. Materials and Methods
2.1. Plant Material and Treatment Layout
The experiment was carried out in the year 2018 in the Italian city of Piacenza (44°55′ N, 9°44′ E) on 20 two-year-old Grechetto Gentile (Vitis vinifera L.) vines (clone VCR433) grown outdoors in 55l pots. These pots were filled with a blend of peat and loamy soil (20:80 by volume). All vines were fertilized on two occasions (that is, one week prior to and two weeks following the bud-break) with 4 grams of Greenplant 15 (N) + 5 (P2O5) + 25 (K2O) + 2 (MgO) + micro. Ten vines each were grafted to 1103 Paulsen rootstock (1103P) and to M4, respectively. After somewhat weak growth recorded during the first year, in winter, each vine was pruned back to one spur with two count nodes. In May, shoot number per vine was standardized by retaining only the two most vertical and robust shoots. Vines were arranged on a vertical shoot, positioned in a 35° NE–SW oriented row, besides being hedgerow-trained with as many as three upper foliage wires for a canopy wall that extended above the graft-union by around 1.8 m. Then, the 20 vines were assigned randomly to four treatments in accordance with the water regime and the rootstock: 1103 Paulsen well-watered (1103P-WW), M4 well-watered (M4-WW), 1103 Paulsen water-stressed (1103P-WS), and M4 water-stressed (M4-WS). Before the commencement of this trial, pots were painted in white in order to curtail overheating induced by radiation.
All the vines were well-watered until DOY 176 (25 June) through the supply of a daily quantity of 6 L in separate fractions at 8:00, 12:00, 15:00, and 18:00. Water stress was imposed only once vines achieved adequate vegetative development (≅ 18–20 unfolded leaves) and in concomitance with high air temperatures and vapor pressure deficit (VPD). Irrigation was withheld in all WS vines until DOY 183 at 18:00 from DOY 177 at 8:00; after that, WS vines were re-watered and complete water supply was maintained for all vines throughout the remainder of the season. During water stress, the pot surface of WS and WW vines was covered using a plastic sheet for preventing infiltration and minimizing losses incurred due to soil evaporation. Shoot trimming was performed on DOY 186 by removing the apical portion of shoots outgrowing 20 cm beyond the top foliage wire.
Daily mean climatic data were derived by a weather station situated in close proximity to the outdoor area, recording hourly rainfall air temperature (T), and relative humidity (RH).
2.2. Gas Exchange Parameters and Chlorophyll Fluorescence
Measurements of leaf gas exchange were recorded daily during the experiment from DOY 176 till DOY 184. More specifically, these readings were undertaken on a mid-shoot mature leaf per vine (five leaves per treatment) under saturating light conditions (PAR > 1400 mmol m–2 s–1) between 12:00 and 13:00 through the utilization of a portable gas exchange LCi infrared gas analyzer (ADC Bio Scientific Ltd., Hertz, UK). Notably, this system was equipped using a wide leaf chamber, having a 6.25 cm2 window. Additionally, all measurements were performed at ambient relative humidity with the adjustment of airflow to 350 mL min–1. Additionally, gas exchanges were measured at 8:00 on DOY 183 and 184, when daytime air temperatures were the lowest, as well as at 18:00, the time the most stressful hours of the afternoon had ended. The calculation of transpiration rate (E), leaf assimilation rate (A), as well as stomatal conductance (gs), was made from concentrations of inlet and outlet CO2 and H2O. The calculation of instantaneous leaf water use efficiency (WUEleaf) was made the ratio between leaf A and leaf E. Percentage loss (PL) of leaf A (PL leaf A) and leaf gs (PL leaf gs) in M4-WS and 1103P-WS was calculated as the daily % difference in leaf A as well as leaf gs vs. the respective WW treatments.
Measurements of chlorophyll fluorescence were conducted on the leaves that were sampled via the field-portable pulse-modulated fluorimeter Handy-PEA (Hansatech Instruments, Norfolk, UK). Segments of leaves were dark-adapted for a period of 30 min via the use of leaf-clips supplied with the instrument. Meanwhile, the fiber optic, as well as its adaptor, were attached to a ring that was situated over the leaf-clip at around 1 cm from the sample, after which varied light pulses were implemented after ensuring compliance with standard routines premised on the user manual’s recommendations.
2.3. Leaf Water Status and Vine Leaf Area
Seasonal progression of water stress was monitored on a daily basis from DOY 177 to 184 through the measurement of leaf pre-dawn water potential (Ψ
pd) prior to sunrise (one leaf per vine; three vines per treatment), along with midday leaf water potential (Ψ
leaf) at 13:00. Additionally, the midday stem water potential (Ψ
stem) on DOY 183 and 184 was measured at the following timings: prior to sunrise, at 8:00, at 13:00, and at 18:00 (one leaf per vine; three vines per treatment). The measurement of Ψ
pd, Ψ
stem, and Ψ
leaf was done on well-exposed and mature basal-medium leaves via a Scholander pressure chamber [
30].
The estimation of the vine leaf area was made at the end of the trial. The leaves that were inserted at nodes 3, 6, 9, 12, and 15 of one shoot per vine were gathered on DOY 186, in conjunction with two corresponding leaves of a lateral that developed below the trimming cut. Each leaf’s area was measured using an LI-3000A leaf area meter (LI-COR Biosciences, Lincoln, NE, USA). Following the leaf fall, the total number of nodes for each cane, as well as for all lateral shoots, were ascertained. Subsequently, an estimation of the final vine leaf area was made from the primary as well as lateral shoots based on node counts and leaf-blade areas.
2.4. Statistical Analysis
A one-way analysis of variance (ANOVA) was undertaken in this study. Additionally, the Student–Newman–Keuls (SNK) test performed the mean separation at p < 0.05, in case of the significance of the F-test. Over time, the data acquired for Ψstem, Ψpd, leaf E, leaf A, Fv/Fm, leaf gs, as well as WUEleaf (denoted as A/E), were assessed using the repeated measure ANOVA routine that forms part of the XLSTAT software package. Mauchly’s sphericity test was utilized for assessing the equality of variances of the differences between all possible pairs of within-subject conditions.
The existing correlations between variables were examined by means of regression analysis through SigmaPlot 11 (Systat Software Inc., San Jose, CA, USA).
4. Discussion
In fully irrigated vines, higher water use from vines grafted on 1103P was quite apparent. It was assessed during the dry down period on both a daily and diurnal basis (DOY 183) and also confirmed upon re-watering (DOY 184). Of course, water use should take into account either transpiration per unit leaf area and the amount of leaf area. It is well known that rootstocks can easily confer different vigor to the grafted scion [
12,
31]; in our study, though, vegetative growth parameters (
Table S1) show that, albeit 1103P had a tendency to slightly push growth, no significant difference was found in vine leaf area at the end of the experiment, therefore re-enforcing the reliability of single leaf readings. A very recent paper by Dayer et al. [
19] had examined changes of key drought-tolerance traits in three cultivars (Grenache, Shiraz, and Semillon) that have largely contrasting water use behavior. Interestingly, they found that the maximum E rate measured under well-watered conditions also affected the variation of a number of drought indices upon progressive water stress. For instance, it was found that increasing maximum E correlated with a lower sensitivity of stomatal conductance to VPD and also with more negative water potential at which stomata close. Transposing these effects to our study only yielded a partial matching. Indeed, higher water used recorded in 1103P-WW vines might have caused faster soil water depletion than in M4, hence also explaining the less rapid decline in pre-dawn and midday leaf water potential. Conversely, in both rootstocks, leaf E rates had a quite similar variation vs. increasing air VPD (not shown) and the same occurred for the Ψ
leaf threshold at which stomatal closure occurred (only −0.03 MPa difference between the two rootstocks vs. about 0.4 MPa difference between Semillon and Grenache according to Dayer et al. [
19]). Thus, it does appear that in our study, grafting Grechetto Gentile vines on either 1103P and M4 did not significantly change the rather conservative behavior of the variety under progressive drought.
However, while the linear model describing the reduction of leaf g
s vs. decreasing Ψ
leaf shared similar slopes and intercepts between the rootstocks (
Figure 4a), when leaf A was taken into account, the linear model fit to the two rootstock data groups still featured the same slope, but the intercept was different (
Figure 4b). Derived conclusions are that, at any given Ψ
leaf, grafting vines on M4 achieves a 15% less limited leaf A, or, reading it the other way, in M4, the Ψ
leaf threshold at which leaf A gets severely limited is reached later in the season.
Meggio et al. [
7], who made a comparison between own-rooted M4 and own-rooted 101−14 Millardet et De Grasset vines, reported higher leaf g
s and leaf A by M4 amidst lower supply of water. SO4 and 101−14 Millardet et De Grasset are classified as weak rootstocks with a low tolerance to water stress [
12,
13,
14,
15]. The novelty of our work is that better performance induced by M4 under moderate-to-severe water stress is also confirmed vs. a drought-tolerant rootstock such as 1103P, therefore broadening its scale of applicability.
Indeed, when it comes to explaining through which mechanisms M4 is able to maintain better leaf function under water stress as compared to other rootstocks, the scenario is still fuzzy. In their experiment on own-rooted potted M4 vines, Meggio et al. hypothesized active osmotic adjustment that was not seen in the commercial rootstock 101.14; from the transcriptomic side, Corso et al. [
16] showed that water-stressed own-rooted M4 vines had higher expression of VsSTS genes coding for resveratrol and flavonoid biosynthesis. The proposed mechanism was that elevated synthesis of resveratrol in M4 roots upon water stress might enhance the ability to cope with oxidative stress usually associated with water deficit [
32]. The last hypothesis has a link with what we found in our work as M4 contributed to counteract photochemical damages to Photosystem 2 under severe WS, as it avoided the permanent reduction of Fv/Fm that was instead recorded in 1103P-WS (
Figure 5).
Differential response of leaf water status and photosynthesis recorded in the two rootstocks translates into higher WUE
leaf under drought for M4. Having a rootstock that can improve WUE
leaf of the grafted scion at moderate to severe water stress levels is very relevant especially in areas where the frequency of significant summer drought is dramatically increasing due to global warming impact. Paradoxically, in the same areas, irrigation is not usually available due to water scarcity or even forbidden due to law-enforced yield limitation. Under such circumstances and in the impossibility of implementing any irrigation strategy, a rootstock that can even slightly enhance vine resilience to water stress by warranting that leaf disruption due to either irreversible photo-inhibition and/or embolism is somewhat postponed is a key factor. In a previous paper from Galbignani et al. [
8], it was shown that whole-canopy water use efficiency was significantly increased in water-stressed potted Sangiovese vines grafted on M4 vs. stressed SO4 when water supply was reduced to 50% and 30% of water lost by the respective well-watered controls. It could be argued that in the present work, we measured WUE
leaf and that, according to literature, the methodology used (i.e., measures taken on single leaves held perpendicular to the sun or under their natural position or directly on the whole canopy) can lead to different conclusions about WUE
leaf variation under water stress [
29,
33,
34]. Medrano et al. [
33] and Poni et al. [
34] have shown, though, that possible mismatch between single leaf or whole canopy derived measurements of WUE
leaf worsens when the whole canopy encompasses large leaf areas and a complex leaf population generating high within-canopy variability in terms leaf age, exposure, and health. This is not the case of our study, where the canopy was given by two vertically growing shoots, in all cases well-exposed to light. Such a simplified canopy makes it very likely that the estimated single WUE
leaf is a good proxy of vine behavior.
Rootstock-induced tolerance to water stress should not be regarded only in terms of maintenance of functionality during a water shortage, rather also as a capacity to induce a prompter resumption of canopy function once non-limiting water availability is replenished in the soil. According to Meggio et al. [
7], own-rooted M4 vines boast a higher leaf A as compared to commercial rootstocks at the restoration of water supply, following the WS period. In our trial, physiological resumption of M4 and 1103P in comparison to the respective WW treatments revealed that M4-WS more promptly recovered leaf gas exchange rates after the complete restoration of water supply. This is another interesting feature of resilience to water stress conferred by the M4 which, quite likely, relates to no significant photo-inhibition experienced by M4 leaves at severe stress.